# Patent

## Title
Method for increasing expression yield of a protein of interest

## Abstract

The present invention relates to a method of producing a protein of interest comprising: (a) cultivating a mutant cell under conditions conducive for production of the protein wherein the mutant has a reduced or no expression of an endogenous polypeptide shown in SEQ ID NO: 2 compared to a parent host cell grown under the same conditions; and optionally (b) recovering the protein of interest.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a 35 U.S.C. 371 national application of PCT/EP2009/052056 filed Feb. 20, 2009, which claims priority or the benefit under 35 U.S.C. 119 of European application no. 08151903.5 filed Feb. 25, 2008 and U.S. provisional application No. 61/031,206 filed Feb. 25, 2008, the contents of which are fully incorporated herein by reference.
REFERENCE TO A SEQUENCE LISTING
This application contains a Sequence Listing in computer readable form. The computer readable form is incorporated herein by reference.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to methods of increasing expression yield of a protein of interest. The present invention in particular relates to isolated polypeptides and isolated polynucleotides encoding polypeptides of the invention. The invention furthermore relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.
2. Description of the Related Art
The use of recombinant host cells in the expression of heterologous proteins has in recent years greatly simplified the production of large quantities of commercially valuable proteins which otherwise are obtainable only by purification from their native sources. Currently, there is a varied selection of expression systems from which to choose for the production of any given protein, including bacterial and eukaryotic hosts. The selection of an appropriate expression system often depends not only on the ability of the host cell to produce adequate yields of the protein in an active state, but, to a large extent, may also be governed by the intended end use of the protein.
Typically improved yields have been obtained by improving the stability of the protein product by using different protease mutants or increasing the expression by using a strong promoter for controlling the expression or improving the secretion pathway by using the most efficient signal peptides.
Host cells used for heterologous protein expression are known to be in a state of stress since the protein synthsizing machinery will be pushed to the limit (Rutkowski, D. T., and Kaufman, R. J. (2004), “A trip to the ER: coping with stress”, Trends in Cell Biology 14 (1): 20-28). Proteins involved in stress responses could therefore be advantageously manipulated leading to improved yield of protein products.
It is an object of the present invention to provide such polypeptides the expressions of which are induced or increased during stress conditions and provide methods for improving yield by modifying the expression.
SUMMARY OF THE INVENTION
The present invention relates to isolated polypeptides selected from the group consisting of:

    
    
        (a) a polypeptide comprising an amino acid sequence having at least 66%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, even more preferably at least 85%, most preferably at least 90%, and even most preferably at least 95% identity to the mature polypeptide of SEQ ID NO: 2;
        (b) a polypeptide encoded by a polynucleotide that hybridizes under at least medium stringency conditions, even more preferably at least medium-high stringency conditions, and most preferably at least high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1, (ii) cDNA sequence contained in the DNA sequence comprising the mature polypeptide coding sequence of SEQ ID NO: 1, or (iii) a full-length complementary strand of (i) or (ii);
        (c) a polypeptide encoded by a polynucleotide comprising a nucleotide sequence having preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, even more preferably at least 85%, most preferably at least 90%, and even most preferably at least 95% identity to the mature polypeptide coding sequence of SEQ ID NO: 1;
        (d) a variant comprising a substitution, deletion, and/or insertion of one or more (several) amino acids of the mature polypeptide of SEQ ID NO: 2.
    
    


The present invention also relates to isolated polynucleotides encoding the polypeptides of the invention.
The present invention also relates to nucleic acid constructs, recombinant expression vectors, recombinant host cells comprising the polynucleotides, and methods of producing a polypeptide of the invention.
The present invention in a further aspect relates to a method of producing the polypeptide of the invention, comprising: (a) cultivating a cell, which in its wild-type form produces the polypeptide, under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.
The present invention in a further aspect relates to a method of producing the polypeptide of the invention, comprising: (a) cultivating a host cell comprising a nucleic acid construct comprising a nucleotide sequence encoding the polypeptide under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.
The present invention also relates to a mutant fungal host, wherein the mutant has a reduced or no expression of an endogenous polypeptide selected from the group consisting of:

    
    
        (a) a polypeptide comprising an amino acid sequence having at least 66%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, even more preferably at least 85%, most preferably at least 90%, and even most preferably at least 95% identity to the mature polypeptide of SEQ ID NO: 2;
        (b) a polypeptide encoded by a polynucleotide that hybridizes under at least medium stringency conditions, even more preferably at least medium-high stringency conditions, and most preferably at least high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1, (ii) cDNA sequence contained in the DNA sequence comprising the mature polypeptide coding sequence of SEQ ID NO: 1, or (iii) a full-length complementary strand of (i) or (ii);
        (c) a polypeptide encoded by a polynucleotide comprising a nucleotide sequence having preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, even more preferably at least 85%, most preferably at least 90%, and even most preferably at least 95% identity to the mature polypeptide coding sequence of SEQ ID NO: 1;
        (d) a polypeptide induced during apoptosis and comprising the motif: EXXCGXXGXMXXDPXXLPEGVXXXXXRXCA; and
        (e) a variant comprising a substitution, deletion, and/or insertion of one or several amino acids of the mature polypeptide of SEQ ID NO: 2.
    
    


The present invention also relates to a method of producing a protein of interest comprising: (a) cultivating a mutant cell under conditions conducive for production of the protein wherein the mutant has a reduced or no expression of an endogenous polypeptide compared to a parent host cell grown under the same conditions, and wherein the expression of said endogenous polypeptide commits the cell to apoptosis; and optionally (b) recovering the protein of interest.
The present invention also relates to methods of producing a protein of interest comprising: (a) cultivating the mutant cell according to the invention further expressing the protein of interest under conditions conducive for production of the protein; and optionally (b) recovering the protein.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 shows the cDNA sequence and the deduced amino acid sequence of Aspergillus oryzae ZY082808 (SEQ ID NOs: 1 and 2, respectively).



DEFINITIONS
Polypeptide induced during apoptosis: The term “expression of said endogenous polypeptide commits the cell to apoptosis” is defined herein to mean that once the endogenous polypeptide has been expressed the host cell will eventually undergo apoptosis.
Apoptosis: The term “apoptosis” is defined herein as cell death resulting from induction of an internal suicide program which prior to the cell death includes cleavage of caspase substrates.
Isolated polypeptide: The term “isolated polypeptide” as used herein refers to a polypeptide that is isolated from a source. In a preferred aspect, the polypeptide is at least 1% pure, preferably at least 5% pure, more preferably at least 10% pure, more preferably at least 20% pure, more preferably at least 40% pure, more preferably at least 60% pure, even more preferably at least 80% pure, and most preferably at least 90% pure, as determined by SDS-PAGE.
Substantially pure polypeptide: The term “substantially pure polypeptide” denotes herein a polypeptide preparation that contains at most 10%, preferably at most 8%, more preferably at most 6%, more preferably at most 5%, more preferably at most 4%, more preferably at most 3%, even more preferably at most 2%, most preferably at most 1%, and even most preferably at most 0.5% by weight of other polypeptide material with which it is natively or recombinantly associated. It is, therefore, preferred that the substantially pure polypeptide is at least 92% pure, preferably at least 94% pure, more preferably at least 95% pure, more preferably at least 96% pure, more preferably at least 96% pure, more preferably at least 97% pure, more preferably at least 98% pure, even more preferably at least 99%, most preferably at least 99.5% pure, and even most preferably 100% pure by weight of the total polypeptide material present in the preparation. The polypeptides of the present invention are preferably in a substantially pure form, i.e., that the polypeptide preparation is essentially free of other polypeptide material with which it is natively or recombinantly associated. This can be accomplished, for example, by preparing the polypeptide by well-known recombinant methods or by classical purification methods.
Mature polypeptide: The term “mature polypeptide” is defined herein as a polypeptide that is in its final form following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. In a preferred aspect, the mature polypeptide is amino acids 20 to 79 of SEQ ID NO: 2 based on the SignalP3.0 program that predicts amino acids 1 to 19 of SEQ ID NO: 2 are a signal peptide.
Mature polypeptide coding sequence: The term “mature polypeptide coding sequence” is defined herein as a nucleotide sequence that encodes a mature polypeptide. In a preferred aspect, the mature polypeptide coding sequence is nucleotides 58 to 237 of SEQ ID NO: 1 based on the SignalP3.0 program that predicts nucleotides 1 to 57 of SEQ ID NO: 1 encode a signal peptide.
Identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter “identity”.
For purposes of the present invention, the degree of identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends in Genetics 16: 276-277), preferably version 3.0.0 or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled “longest identity” (obtained using the—nobrief option) is used as the percent identity and is calculated as follows:

(Identical Residues×100)/(Length of Alignment−Total Number of Gaps in Alignment)

For purposes of the present invention, the degree of identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 3.0.0 or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled “longest identity” (obtained using the—nobrief option) is used as the percent identity and is calculated as follows:

(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Number of Gaps in Alignment)

Homologous sequence: The term “homologous sequence” is defined herein as a predicted protein that gives an E value (or expectancy score) of less than 0.001 in a tfasty search (Pearson, W. R., 1999, in Bioinformatics Methods and Protocols, S. Misener and S. A. Krawetz, ed., pp. 185-219) with the Aspergillus oryzae ZY082808 polypeptide of SEQ ID NO: 2.
Polypeptide fragment: The term “polypeptide fragment” is defined herein as a polypeptide having one or more (several) amino acids deleted from the amino and/or carboxyl terminus of the mature polypeptide of SEQ ID NO: 2; wherein the fragment has retained its activity.
Subsequence: The term “subsequence” is defined herein as a nucleotide sequence having one or more (several) nucleotides deleted from the 5′ and/or 3′ end of the mature polypeptide coding sequence of SEQ ID NO: 1; wherein the subsequence encodes a polypeptide fragment having retained its activity.
Allelic variant: The term “allelic variant” denotes herein any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequences. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.
Isolated polynucleotide: The term “isolated polynucleotide” as used herein refers to a polynucleotide that is isolated from a source. In a preferred aspect, the polynucleotide is at least 1% pure, preferably at least 5% pure, more preferably at least 10% pure, more preferably at least 20% pure, more preferably at least 40% pure, more preferably at least 60% pure, even more preferably at least 80% pure, and most preferably at least 90% pure, as determined by agarose electrophoresis.
Substantially pure polynucleotide: The term “substantially pure polynucleotide” as used herein refers to a polynucleotide preparation free of other extraneous or unwanted nucleotides and in a form suitable for use within genetically engineered protein production systems. Thus, a substantially pure polynucleotide contains at most 10%, preferably at most 8%, more preferably at most 6%, more preferably at most 5%, more preferably at most 4%, more preferably at most 3%, even more preferably at most 2%, most preferably at most 1%, and even most preferably at most 0.5% by weight of other polynucleotide material with which it is natively or recombinantly associated. A substantially pure polynucleotide may, however, include naturally occurring 5′ and 3′ untranslated regions, such as promoters and terminators. It is preferred that the substantially pure polynucleotide is at least 90% pure, preferably at least 92% pure, more preferably at least 94% pure, more preferably at least 95% pure, more preferably at least 96% pure, more preferably at least 97% pure, even more preferably at least 98% pure, most preferably at least 99%, and even most preferably at least 99.5% pure by weight. The polynucleotides of the present invention are preferably in a substantially pure form, i.e., that the polynucleotide preparation is essentially free of other polynucleotide material with which it is natively or recombinantly associated. The polynucleotides may be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or any combinations thereof.
Coding sequence: When used herein the term “coding sequence” means a nucleotide sequence, which directly specifies the amino acid sequence of its protein product. The boundaries of the coding sequence are generally determined by an open reading frame, which usually begins with the ATG start codon or alternative start codons such as GTG and TTG and ends with a stop codon such as TAA, TAG, and TGA. The coding sequence may be a DNA, cDNA, synthetic, or recombinant nucleotide sequence.
cDNA: The term “cDNA” is defined herein as a DNA molecule that can be prepared by reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic cell. cDNA lacks intron sequences that are usually present in the corresponding genomic DNA. The initial, primary RNA transcript is a precursor to mRNA that is processed through a series of steps before appearing as mature spliced mRNA. These steps include the removal of intron sequences by a process called splicing. cDNA derived from mRNA lacks, therefore, any intron sequences.
Nucleic acid construct: The term “nucleic acid construct” as used herein refers to a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or which is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic. The term nucleic acid construct is synonymous with the term “expression cassette” when the nucleic acid construct contains the control sequences required for expression of a coding sequence of the present invention.
Control sequences: The term “control sequences” is defined herein to include all components necessary for the expression of a polynucleotide encoding a polypeptide of the present invention. Each control sequence may be native or foreign to the nucleotide sequence encoding the polypeptide or native or foreign to each other. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleotide sequence encoding a polypeptide.
Operably linked: The term “operably linked” denotes herein a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of the polynucleotide sequence such that the control sequence directs the expression of the coding sequence of a polypeptide.
Expression: The term “expression” includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
Expression vector: The term “expression vector” is defined herein as a linear or circular DNA molecule that comprises a polynucleotide encoding a polypeptide of the present invention and is operably linked to additional nucleotides that provide for its expression.
Host cell: The term “host cell”, as used herein, includes any cell type that is susceptible to transformation, transfection, transduction, and the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention.
Modification: The term “modification” means herein any chemical modification of the polypeptide consisting of the mature polypeptide of SEQ ID NO: 2; or a homologous sequence thereof; as well as genetic manipulation of the DNA encoding such a polypeptide. The modification can be a substitution, a deletion and/or an insertion of one or more (several) amino acids as well as replacements of one or more (several) amino acid side chains.
DETAILED DESCRIPTION OF THE INVENTION
When fungi are used as expression host cells for the production of proteins the protein of interest should be produced in a sufficient amount. As discussed briefly above in order to improve yield of such proteins several factors have traditionally been studied. Host cells used in the production of such proteins, which could e.g. be a heterologous protein expressed recombinantly in the host or an endogenous protein produced in high quantities, will be in a stress situation (Rutkowski, D. T., and Kaufman, R. J. (2004), “A trip to the ER: coping with stress”, Trends in Cell Biology 14 (1): 20-28). In the present invention expression yield have been improved by affecting the expression of specific factors the expression of which seems to be influenced by the level of stress the host cell is subjected to. Such factors have been identified using a cDNA library of A. oryzae expressed sequence tags (ESTs) in a DNA array. A number of genes were differentially regulated and among these the polypeptide according to the present invention.
Polypeptides of the Invention
In a first aspect, the present invention relates to isolated polypeptides comprising an amino acid sequence having a degree of identity to the mature polypeptide of SEQ ID NO: 2 of preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 96%, at least 97%, at least 98%, or at least 99% (hereinafter “homologous polypeptides”). In a preferred aspect, the homologous polypeptides have an amino acid sequence that differs by ten amino acids, preferably by five amino acids, more preferably by four amino acids, even more preferably by three amino acids, most preferably by two amino acids, and even most preferably by one amino acid from the mature polypeptide of SEQ ID NO: 2.
A polypeptide of the present invention preferably comprises the amino acid sequence of SEQ ID NO: 2 or an allelic variant thereof; or a fragment thereof. In a preferred aspect, the polypeptide comprises the amino acid sequence of SEQ ID NO: 2. In another preferred aspect, the polypeptide comprises the mature polypeptide of SEQ ID NO: 2. In another preferred aspect, the polypeptide comprises amino acids 20 to 79 of SEQ ID NO: 2, or an allelic variant thereof; or a fragment thereof. In another preferred aspect, the polypeptide comprises amino acids 20 to 79 of SEQ ID NO: 2. In another preferred aspect, the polypeptide consists of the amino acid sequence of SEQ ID NO: 2 or an allelic variant thereof; or a fragment thereof. In another preferred aspect, the polypeptide consists of the amino acid sequence of SEQ ID NO: 2. In another preferred aspect, the polypeptide consists of the mature polypeptide of SEQ ID NO: 2. In another preferred aspect, the polypeptide consists of amino acids 20 to 79 of SEQ ID NO: 2 or an allelic variant thereof; or a fragment thereof. In another preferred aspect, the polypeptide consists of amino acids 20 to 79 of SEQ ID NO: 2.
In a second aspect, the present invention relates to isolated polypeptides that are encoded by polynucleotides that hybridize under preferably very low stringency conditions, more preferably low stringency conditions, more preferably medium stringency conditions, more preferably medium-high stringency conditions, even more preferably high stringency conditions, and most preferably very high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1, (ii) the cDNA sequence contained in SEQ ID NO: 1, (iii) a subsequence of (i) or (ii), or (iv) a full-length complementary strand of (i), (ii), or (iii) (J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, N.Y.). A subsequence of the mature polypeptide coding sequence of SEQ ID NO: 1 contains at least 100 contiguous nucleotides or preferably at least 200 contiguous nucleotides. In a preferred aspect, the complementary strand is the full-length complementary strand of the mature polypeptide coding sequence of SEQ ID NO: 1.
The nucleotide sequence of SEQ ID NO: 1; or a subsequence thereof; as well as the amino acid sequence of SEQ ID NO: 2; or a fragment thereof; may be used to design nucleic acid probes to identify and clone DNA encoding polypeptides from strains of different genera or species according to methods well known in the art. In particular, such probes can be used for hybridization with the genomic or cDNA of the genus or species of interest, following standard Southern blotting procedures, in order to identify and isolate the corresponding gene therein. Such probes can be considerably shorter than the entire sequence, but should be at least 14, preferably at least 25, more preferably at least 35, and most preferably at least 70 nucleotides in length. It is, however, preferred that the nucleic acid probe is at least 100 nucleotides in length. For example, the nucleic acid probe may be at least 200 nucleotides, preferably at least 300 nucleotides, more preferably at least 400 nucleotides, or most preferably at least 500 nucleotides in length. Both DNA and RNA probes can be used. The probes are typically labeled for detecting the corresponding gene (for example, with 32P, 3H, 35S, biotin, or avidin). Such probes are encompassed by the present invention.
A genomic DNA or cDNA library prepared from such other strains may, therefore, be screened for DNA that hybridizes with the probes described above and encodes a functionally homologous polypeptide. Genomic or other DNA from such other strains may be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques. DNA from the libraries or the separated DNA may be transferred to and immobilized on nitrocellulose or other suitable carrier material. In order to identify a clone or DNA that is homologous with SEQ ID NO: 1; or a subsequence thereof; the carrier material is preferably used in a Southern blot.
For purposes of the present invention, hybridization indicates that the nucleotide sequence hybridizes to a labeled nucleic acid probe corresponding to the mature polypeptide coding sequence of SEQ ID NO: 1; the cDNA sequence contained in SEQ ID NO: 1; its full-length complementary strand; or a subsequence thereof; under very low to very high stringency conditions. Molecules to which the nucleic acid probe hybridizes under these conditions can be detected using, for example, X-ray film.
In a preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 1. In another preferred aspect, the nucleic acid probe is nucleotides 58 to 237 of SEQ ID NO: 1. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 2, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 1.
For long probes of at least 100 nucleotides in length, very low to very high stringency conditions are defined as prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA, and either 25% formamide for very low and low stringencies, 35% formamide for medium and medium-high stringencies, or 50% formamide for high and very high stringencies, following standard Southern blotting procedures for 12 to 24 hours optimally.
For long probes of at least 100 nucleotides in length, the carrier material is finally washed three times each for 15 minutes using 2×SSC, 0.2% SDS preferably at 45° C. (very low stringency), more preferably at 50° C. (low stringency), more preferably at 55° C. (medium stringency), more preferably at 60° C. (medium-high stringency), even more preferably at 65° C. (high stringency), and most preferably at 70° C. (very high stringency).
For short probes that are about 15 nucleotides to about 70 nucleotides in length, stringency conditions are defined as prehybridization, hybridization, and washing post-hybridization at about 5° C. to about 10° C. below the calculated Tm using the calculation according to Bolton and McCarthy (1962, Proceedings of the National Academy of Sciences USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA, 0.5% NP-40, 1× Denhardt's solution, 1 mM sodium pyrophosphate, 1 mM sodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per ml following standard Southern blotting procedures for 12 to 24 hours optimally.
For short probes that are about 15 nucleotides to about 70 nucleotides in length, the carrier material is washed once in 6×SCC plus 0.1% SDS for 15 minutes and twice each for 15 minutes using 6×SSC at 5° C. to 10° C. below the calculated Tm.
In a third aspect, the present invention relates to isolated polypeptides encoded by polynucleotides comprising or consisting of nucleotide sequences that have a degree of identity to the mature polypeptide coding sequence of SEQ ID NO: 1 of preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably 96%, 97%, 98%, or 99%, which encode an active polypeptide. See polynucleotide section herein.
In another aspect, the the isolated polypeptides comprise the following motif:
EXXCGXXGXMXXDPXXLPEGVXXXXXRXCA,
wherein x is any amino acid. In the above motif, the accepted IUPAC single letter amino acid abbreviation is employed.
In a still further aspect, the present invention relates to artificial variants comprising a substitution, deletion, and/or insertion of one or more (or several) amino acids of the mature polypeptide of SEQ ID NO: 2; or a homologous polypeptide thereof. Preferably, amino acid changes are of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of one to about 30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to about 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope or a binding domain.
Examples of conservative substitutions are within the group of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions that do not generally alter specific activity are known in the art and are described, for example, by H. Neurath and R. L. Hill, 1979, In, The Proteins, Academic Press, New York. The most commonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, LeuNal, Ala/Glu, and Asp/Gly.
In addition to the 20 standard amino acids, non-standard amino acids (such as 4-hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid, isovaline, and alpha-methyl serine) may be substituted for amino acid residues of a wild-type polypeptide. A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, and unnatural amino acids may be substituted for amino acid residues. “Unnatural amino acids” have been modified after protein synthesis, and/or have a chemical structure in their side chain(s) different from that of the standard amino acids. Unnatural amino acids can be chemically synthesized, and preferably, are commercially available, and include pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline, and 3,3-dimethylproline.
Alternatively, the amino acid changes are of such a nature that the physico-chemical properties of the polypeptides are altered. For example, amino acid changes may improve the thermal stability of the polypeptide, alter the substrate specificity, change the pH optimum, and the like.
Essential amino acids in the parent polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The active site of the enzyme or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identities of essential amino acids can also be inferred from analysis of identities with polypeptides that are related to a polypeptide according to the invention.
Single or multiple amino acid substitutions, deletions, and/or insertions can be made and tested using known methods of mutagenesis, recombination, and/or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochem. 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).
Mutagenesis/shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure.
The total number of amino acid substitutions, deletions and/or insertions of the mature polypeptide of SEQ ID NO: 2, such as amino acids 20 to 79 of SEQ ID NO: 2, is 10, preferably 9, more preferably 8, more preferably 7, more preferably at most 6, more preferably 5, more preferably 4, even more preferably 3, most preferably 2, and even most preferably 1.
Sources of Polypeptides
A polypeptide of the present invention may be obtained from microorganisms of any genus. For purposes of the present invention, the term “obtained from” as used herein in connection with a given source shall mean that the polypeptide encoded by a nucleotide sequence is produced by the source or by a strain in which the nucleotide sequence from the source has been inserted. In a preferred aspect, the polypeptide obtained from a given source is secreted extracellularly.
A polypeptide of the present invention the expression of which commits the host cell to apoptosis may be a fungal polypeptide, and more preferably a yeast polypeptide such as a Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia polypeptide; or more preferably a filamentous fungal polypeptide such as an Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium, Botryospaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, lrpex, Lentinula, Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Piromyces, Poitrasia, Pseudoplectania, Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvariella, or Xylaria polypeptide.
In a preferred aspect, the polypeptide is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, or Saccharomyces oviformis polypeptide.
In another preferred aspect, the polypeptide is an Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium tropicum, Chrysosporium merdarium, Chrysosporium inops, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium zonatum, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa, Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaete chrysosporium, Thielavia achromatica, Thielavia albomyces, Thielavia albopilosa, Thielavia australeinsis, Thielavia fimeti, Thielavia microspora, Thielavia ovispora, Thielavia peruviana, Thielavia spededonium, Thielavia setosa, Thielavia subthermophila, Thielavia terrestris, Trichoderma harzianum, Trichoderma koningii, Trichoderma Iongibrachiatum, Trichoderma reesei, or Trichoderma viride polypeptide.
In another preferred aspect, the polypeptide is an Aspergillus oryzae, Aspergillus niger, polypeptide.
In a more preferred aspect, the polypeptide is an Aspergillus oryzae polypeptide. In a most preferred aspect, the polypeptide is an Aspergillus oryzae A1560 (NBRC4177) polypeptide, e.g., the polypeptide comprising the mature polypeptide of SEQ ID NO: 2.
It will be understood that for the aforementioned species the invention encompasses both the perfect and imperfect states, and other taxonomic equivalents, e.g., anamorphs, regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents.
Strains of these species are readily accessible to the public in a number of culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).
Furthermore, such polypeptides may be identified and obtained from other sources including microorganisms isolated from nature (e.g., soil, composts, water, etc.) using the above-mentioned probes. Techniques for isolating microorganisms from natural habitats are well known in the art. The polynucleotide may then be obtained by similarly screening a genomic or cDNA library of such a microorganism. Once a polynucleotide sequence encoding a polypeptide has been detected with the probe(s), the polynucleotide can be isolated or cloned by utilizing techniques that are well known to those of ordinary skill in the art (see, e.g., Sambrook et al., 1989, supra).
Polypeptides of the present invention also include fused polypeptides or cleavable fusion polypeptides in which another polypeptide is fused at the N-terminus or the C-terminus of the polypeptide or fragment thereof. A fused polypeptide is produced by fusing a nucleotide sequence (or a portion thereof) encoding another polypeptide to a nucleotide sequence (or a portion thereof) of the present invention. Techniques for producing fusion polypeptides are known in the art, and include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fused polypeptide is under control of the same promoter(s) and terminator.
A fusion polypeptide can further comprise a cleavage site. Upon secretion of the fusion protein, the site is cleaved releasing the polypeptide of the invention from the fusion protein. Examples of cleavage sites include, but are not limited to, a Kex2 site that encodes the dipeptide Lys-Arg (Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3: 568-76; Svetina et al., 2000, J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13: 498-503; and Contreras et al., 1991, Biotechnology 9: 378-381), an Ile-(Glu or Asp)-Gly-Arg site, which is cleaved by a Factor Xa protease after the arginine residue (Eaton et al., 1986, Biochem. 25: 505-512); a Asp-Asp-Asp-Asp-Lys site, which is cleaved by an enterokinase after the lysine (Collins-Racie et al., 1995, Biotechnology 13: 982-987); a His-Tyr-Glu site or His-Tyr-Asp site, which is cleaved by Genenase I (Carter et al., 1989, Proteins: Structure, Function, and Genetics 6: 240-248); a Leu-Val-Pro-Arg-Gly-Ser site, which is cleaved by thrombin after the Arg (Stevens, 2003, Drug Discovery World 4: 35-48); a Glu-Asn-Leu-Tyr-Phe-Gln-Gly site, which is cleaved by TEV protease after the Gln (Stevens, 2003, supra); and a Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro site, which is cleaved by a genetically engineered form of human rhinovirus 3C protease after the Gln (Stevens, 2003, supra).
Polynucleotides
The present invention also relates to isolated polynucleotides comprising or consisting of nucleotide sequences that encode polypeptides the expression of which commits the host cell to apoptosis of the present invention.
In a preferred aspect, the nucleotide sequence comprises or consists of SEQ ID NO: 1. In another preferred aspect, the nucleotide sequence comprises or consists of the mature polypeptide coding sequence of SEQ ID NO: 1. In another preferred aspect, the nucleotide sequence comprises or consists of nucleotides 58 to 237 of SEQ ID NO: 1. The present invention also encompasses nucleotide sequences that encode polypeptides comprising or consisting of the amino acid sequence of SEQ ID NO: 2 or the mature polypeptide thereof, which differ from SEQ ID NO: 1 or the mature polypeptide coding sequence thereof by virtue of the degeneracy of the genetic code. The present invention also relates to subsequences of SEQ ID NO: 1 that encode fragments of SEQ ID NO: 2.
The present invention also relates to mutant polynucleotides comprising or consisting of at least one mutation in the mature polypeptide coding sequence of SEQ ID NO: 1, in which the mutant nucleotide sequence encodes the mature polypeptide of SEQ ID NO: 2.
The techniques used to isolate or clone a polynucleotide encoding a polypeptide are known in the art and include isolation from genomic DNA, preparation from cDNA, or a combination thereof. The cloning of the polynucleotides of the present invention from such genomic DNA can be effected, e.g., by using the well known polymerase chain reaction (PCR) or antibody screening of expression libraries to detect cloned DNA fragments with shared structural features. See, e.g., Innis et al., 1990, PCR: A Guide to Methods and Application, Academic Press, New York. Other nucleic acid amplification procedures such as ligase chain reaction (LCR), ligated activated transcription (LAT) and nucleotide sequence-based amplification (NASBA) may be used. The polynucleotides may be cloned from a strain of Aspergillus, or another or related organism and thus, for example, may be an allelic or species variant of the polypeptide encoding region of the nucleotide sequence.
The present invention also relates to isolated polynucleotides comprising or consisting of nucleotide sequences that have a degree of identity to the mature polypeptide coding sequence of SEQ ID NO: 1 of preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 96%, at least 97%, at least 98%, or at least 99% identity, which encode an active polypeptide.
Modification of a nucleotide sequence encoding a polypeptide of the present invention may be necessary for the synthesis of polypeptides substantially similar to the polypeptide. The term “substantially similar” to the polypeptide refers to non-naturally occurring forms of the polypeptide. These polypeptides may differ in some engineered way from the polypeptide isolated from its native source, e.g., artificial variants that differ in specific activity, thermostability, pH optimum, or the like. The variant sequence may be constructed on the basis of the nucleotide sequence presented as the mature polypeptide coding sequence of SEQ ID NO: 1, e.g., a subsequence thereof, and/or by introduction of nucleotide substitutions that do not give rise to another amino acid sequence of the polypeptide encoded by the nucleotide sequence, but which correspond to the codon usage of the host organism intended for production of the enzyme, or by introduction of nucleotide substitutions that may give rise to a different amino acid sequence. For a general description of nucleotide substitution, see, e.g., Ford et al., 1991, Protein Expression and Purification 2: 95-107.
It will be apparent to those skilled in the art that such substitutions can be made outside the regions critical to the function of the molecule and still result in an active polypeptide. Amino acid residues essential to the activity of the polypeptide encoded by an isolated polynucleotide of the invention, and therefore preferably not subject to substitution, may be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (see, e.g., Cunningham and Wells, 1989, supra). In the latter technique, mutations are introduced at every positively charged residue in the molecule, and the resultant mutant molecules are tested for maintained activity to identify amino acid residues that are critical to the activity of the molecule. Sites of substrate-enzyme interaction can also be determined by analysis of the three-dimensional structure as determined by such techniques as nuclear magnetic resonance analysis, crystallography or photoaffinity labeling (see, e.g., de Vos et al., 1992, supra; Smith et al., 1992, supra; Wlodaver et al., 1992, supra).
The present invention also relates to isolated polynucleotides encoding polypeptides of the present invention, which hybridize under very low stringency conditions, preferably low stringency conditions, more preferably medium stringency conditions, more preferably medium-high stringency conditions, even more preferably high stringency conditions, and most preferably very high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1, (ii) the cDNA sequence contained in SEQ ID NO: 1, or (iii) a full-length complementary strand of (i) or (ii); or allelic variants and subsequences thereof (Sambrook et al., 1989, supra), as defined herein. In a preferred aspect, the complementary strand is the full-length complementary strand of the mature polypeptide coding sequence of SEQ ID NO: 1.
The present invention also relates to isolated polynucleotides obtained by (a) hybridizing a population of DNA under very low, low, medium, medium-high, high, or very high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1, (ii) the cDNA sequence contained SEQ ID NO: 1, or (iii) a full-length complementary strand of (i) or (ii); and (b) isolating the hybridizing polynucleotide, which encodes a polypeptide the expression of which commits the host cell to apoptosis. In a preferred aspect, the complementary strand is the full-length complementary strand of the mature polypeptide coding sequence of SEQ ID NO: 1.
Nucleic Acid Constructs
The present invention also relates to nucleic acid constructs comprising an isolated polynucleotide of the present invention operably linked to one or more (several) control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.
An isolated polynucleotide encoding a polypeptide of the present invention may be manipulated in a variety of ways to provide for expression of the polypeptide. Manipulation of the polynucleotide's sequence prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotide sequences utilizing recombinant DNA methods are well known in the art.
The control sequence may be an appropriate promoter sequence, a nucleotide sequence that is recognized by a host cell for expression of a polynucleotide encoding a polypeptide of the present invention. The promoter sequence contains transcriptional control sequences that mediate the expression of the polypeptide. The promoter may be any nucleotide sequence that shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.
Examples of suitable promoters for directing the transcription of the nucleic acid constructs of the present invention in a filamentous fungal host cell are promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, Fusarium venenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO 00/56900), Fusarium venenatum Quinn (WO 00/56900), Fusarium oxysporum trypsin-like protease (WO 96/00787), Trichoderma reesei beta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase IV, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpi promoter (a hybrid of the promoters from the genes for Aspergillus niger neutral alpha-amylase and Aspergillus oryzae triose phosphate isomerase); and mutant, truncated, and hybrid promoters thereof.
In a yeast host, useful promoters are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP), Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomyces cerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are described by Romanos et al., 1992, Yeast 8: 423-488.
The control sequence may also be a suitable transcription terminator sequence, a sequence recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3′ terminus of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the host cell of choice may be used in the present invention.
Preferred terminators for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease.
Preferred terminators for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described by Romanos et al., 1992, supra.
The control sequence may also be a suitable leader sequence, a nontranslated region of an mRNA that is important for translation by the host cell. The leader sequence is operably linked to the 5′ terminus of the nucleotide sequence encoding the polypeptide. Any leader sequence that is functional in the host cell of choice may be used in the present invention.
Preferred leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.
Suitable leaders for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).
The control sequence may also be a polyadenylation sequence; a sequence operably linked to the 3′ terminus of the nucleotide sequence and, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence that is functional in the host cell of choice may be used in the present invention.
Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger alpha-glucosidase.
Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Molecular Cellular Biology 15: 5983-5990.
The control sequence may also be a signal peptide coding sequence that codes for an amino acid sequence linked to the amino terminus of a polypeptide and directs the encoded polypeptide into the cell's secretory pathway. The 5′ end of the coding sequence of the nucleotide sequence may inherently contain a signal peptide coding sequence naturally linked in translation reading frame with the segment of the coding sequence that encodes the secreted polypeptide. Alternatively, the 5′ end of the coding sequence may contain a signal peptide coding sequence that is foreign to the coding sequence. The foreign signal peptide coding sequence may be required where the coding sequence does not naturally contain a signal peptide coding sequence. Alternatively, the foreign signal peptide coding sequence may simply replace the natural signal peptide coding sequence in order to enhance secretion of the polypeptide. However, any signal peptide coding sequence that directs the expressed polypeptide into the secretory pathway of a host cell of choice, i.e., secreted into a culture medium, may be used in the present invention.
Effective signal peptide coding sequences for filamentous fungal host cells are the signal peptide coding sequences obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, Humicola insolens endoglucanase V, and Humicola lanuginosa lipase.
Useful signal peptides for yeast host cells are obtained from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding sequences are described by Romanos et al., 1992, supra.
In one embodiment the signal peptide is the normal signal associated with the polypeptide of the invention.
In one aspect, the signal peptide comprises or consists of amino acids 1 to 19 of SEQ ID NO: 2. In another aspect, the signal peptide coding sequence comprises or consists of nucleotides 1 to 57 of SEQ ID NO: 1.
The control sequence may also be a propeptide coding sequence that codes for an amino acid sequence positioned at the amino terminus of a polypeptide. The resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases). A propeptide is generally inactive and can be converted to a mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding sequence may be obtained from the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiae alpha-factor, Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophila laccase (WO 95/33836).
Where both signal peptide and propeptide sequences are present at the amino terminus of a polypeptide, the propeptide sequence is positioned next to the amino terminus of a polypeptide and the signal peptide sequence is positioned next to the amino terminus of the propeptide sequence.
It may also be desirable to add regulatory sequences that allow the regulation of the expression of the polypeptide relative to the growth of the host cell. Examples of regulatory systems are those that cause the expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Regulatory systems in prokaryotic systems include the lac, tac, xyl and trp operator systems. In yeast, the ADH2 system or GAL1 system may be used. In filamentous fungi, the TAKA alpha-amylase promoter, Aspergillus niger glucoamylase promoter, and Aspergillus oryzae glucoamylase promoter may be used as regulatory sequences. Other examples of regulatory sequences are those that allow for gene amplification. In eukaryotic systems, these regulatory sequences include the dihydrofolate reductase gene that is amplified in the presence of methotrexate, and the metallothionein genes that are amplified with heavy metals. In these cases, the nucleotide sequence encoding the polypeptide would be operably linked with the regulatory sequence.
Expression Vectors
The present invention also relates to recombinant expression vectors comprising a polynucleotide of the present invention, a promoter, and transcriptional and translational stop signals. The various nucleic acids and control sequences described herein may be joined together to produce a recombinant expression vector that may include one or more (several) convenient restriction sites to allow for insertion or substitution of the nucleotide sequence encoding the polypeptide at such sites. Alternatively, a polynucleotide sequence of the present invention may be expressed by inserting the nucleotide sequence or a nucleic acid construct comprising the sequence into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.
The recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of the nucleotide sequence. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vectors may be linear or closed circular plasmids.
The vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon, may be used.
The vectors of the present invention preferably contain one or more (several) selectable markers that permit easy selection of transformed, transfected, transduced, or the like cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.
Examples of bacterial selectable markers are the dal genes from Bacillus subtilis or Bacillus licheniformis, or markers that confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol, or tetracycline resistance. Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungal host cell include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof. Preferred for use in an Aspergillus cell are the amdS and pyrG genes of Aspergillus nidulans or Aspergillus oryzae and the bargene of Streptomyces hygroscopicus. 
The vectors of the present invention preferably contain an element(s) that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.
For integration into the host cell genome, the vector may rely on the polynucleotide's sequence encoding the polypeptide or any other element of the vector for integration into the genome by homologous or nonhomologous recombination. Alternatively, the vector may contain additional nucleotide sequences for directing integration by homologous recombination into the genome of the host cell at a precise location(s) in the chromosome(s). To increase the likelihood of integration at a precise location, the integrational elements should preferably contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000 base pairs, which have a high degree of identity to the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, the integrational elements may be non-encoding or encoding nucleotide sequences. On the other hand, the vector may be integrated into the genome of the host cell by non-homologous recombination.
For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replicator mediating autonomous replication that functions in a cell. The term “origin of replication” or “plasmid replicator” is defined herein as a nucleotide sequence that enables a plasmid or vector to replicate in vivo.
Examples of origins of replication for use in a yeast host cell are the 2 micron origin of replication, ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6.
Examples of origins of replication useful in a filamentous fungal cell are AMA1 and ANS1 (Gems et al., 1991, Gene 98: 61-67; Cullen et al., 1987, Nucleic Acids Research 15: 9163-9175; WO 00/24883). Isolation of the AMA1 gene and construction of plasmids or vectors comprising the gene can be accomplished according to the methods disclosed in WO 00/24883.
More than one copy of a polynucleotide of the present invention may be inserted into a host cell to increase production of the gene product. An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the polynucleotide, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.
The procedures used to ligate the elements described above to construct the recombinant expression vectors of the present invention are well known to one skilled in the art (see, e.g., Sambrook et al., 1989, supra).
Host Cells
The present invention also relates to recombinant host cells, comprising an isolated polynucleotide of the present invention, which are advantageously used in the recombinant production of the polypeptides. A vector comprising a polynucleotide of the present invention is introduced into a host cell so that the vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector as described earlier. The term “host cell” encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of a host cell will to a large extent depend upon the gene encoding the polypeptide and its source.
The host cell may be any eukaryote, such as a mammalian, insect, plant, or fungal cell.
In a preferred aspect, the host cell is a fungal cell. “Fungi” as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota (as defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK) as well as the Oomycota (as cited in Hawksworth et al., 1995, supra, page 171) and all mitosporic fungi (Hawksworth et al., 1995, supra).
In a more preferred aspect, the fungal host cell is a yeast cell. “Yeast” as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes). Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, F. A., Passmore, S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium Series No. 9, 1980).
In an even more preferred aspect, the yeast host cell is a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell.
In a most preferred aspect, the yeast host cell is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, or Saccharomyces oviformis cell. In another most preferred aspect, the yeast host cell is a Kluyveromyces lactis cell. In another most preferred aspect, the yeast host cell is a Yarrowia lipolytica cell.
In another more preferred aspect, the fungal host cell is a filamentous fungal cell. “Filamentous fungi” include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra). The filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon catabolism may be fermentative.
In an even more preferred aspect, the filamentous fungal host cell is an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell.
In a most preferred aspect, the filamentous fungal host cell is an Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger or Aspergillus oryzae cell. In another most preferred aspect, the filamentous fungal host cell is a Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusarium venenatum cell. In another most preferred aspect, the filamentous fungal host cell is a Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium tropicum, Chrysosporium merdarium, Chrysosporium inops, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.
Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transformation of Aspergillus and Trichoderma host cells are described in EP 238 023 and Yelton et al., 1984, Proceedings of the National Academy of Sciences USA 81: 1470-1474. Suitable methods for transforming Fusarium species are described by Malardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Ito et al., 1983, Journal of Bacteriology 153: 163; and Hinnen et al., 1978, Proceedings of the National Academy of Sciences USA 75: 1920.
Methods of Production
The present invention also relates to methods of producing a polypeptide of the present invention, comprising: (a) cultivating a cell, which in its wild-type form produces the polypeptide, under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide. In a preferred aspect, the cell is of the genus Aspergillus. In a more preferred aspect, the cell is Aspergillus oryzae. 
The present invention also relates to methods of producing a polypeptide of the present invention, comprising: (a) cultivating a recombinant host cell, as described herein, under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.
In the production methods of the present invention, the cells are cultivated in a nutrient medium suitable for production of the polypeptide using methods well known in the art. For example, the cell may be cultivated by shake flask cultivation, and small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the polypeptide to be expressed and/or isolated. The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted into the medium, it can be recovered from cell lysates.
The polypeptides may be detected using methods known in the art that are specific for the polypeptides. These detection methods may include use of specific antibodies, formation of an enzyme product, or disappearance of an enzyme substrate. For example, an enzyme assay may be used to determine the activity of the polypeptide as described herein.
The resulting polypeptide may be recovered using methods known in the art. For example, the polypeptide may be recovered from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation.
The polypeptides of the present invention may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989) to obtain substantially pure polypeptides.
Removal or Reduction of ZY082808 Activity
The present invention also relates to a mutant of a parent cell, which comprises modifying, disrupting or deleting a polynucleotide sequence, or a portion thereof, encoding a polypeptide of the present invention, which results in the mutant cell producing less of the polypeptide than the parent cell when cultivated under the same conditions.
The mutant cell may be constructed by reducing or eliminating expression of a nucleotide sequence encoding a polypeptide of the present invention using methods well known in the art, for example, insertions, disruptions, replacements, or deletions. In a preferred aspect, the nucleotide sequence is modified so that the encoded polypeptide is inactivated. The nucleotide sequence to be modified may be, for example, the coding region or a part thereof essential for activity, or a regulatory element required for the expression of the coding region. An example of such a regulatory or control sequence may be a promoter sequence or a functional part thereof, i.e., a part that is sufficient for affecting expression of the nucleotide sequence. Other control sequences for possible modification include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, signal peptide sequence, transcription terminator, and transcriptional activator.
Thus in one embodiment the present invention relates to a mutant fungal host, wherein the mutant has a reduced or no expression of an endogenous polypeptide selected from the group consisiting of:
(a) a polypeptide comprising an amino acid sequence having at least 66%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, even more preferably at least 85%, most preferably at least 90%, and even most preferably at least 95% identity to the mature polypeptide of SEQ ID NO: 2;
(b) a polypeptide encoded by a polynucleotide that hybridizes under at least medium stringency conditions, even more preferably at least medium-high stringency conditions, and most preferably at least high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1, (ii) cDNA sequence contained in the DNA sequence comprising the mature polypeptide coding sequence of SEQ ID NO: 1, or (iii) a full-length complementary strand of (i) or (ii);
(c) a polypeptide encoded by a polynucleotide comprising a nucleotide sequence having [Edit accordingly] preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, even more preferably at least 85%, most preferably at least 90%, and even most preferably at least 95% identity to the mature polypeptide coding sequence of SEQ ID NO: 1;
(d) a polypeptide induced during apoptosis and comprising the motif: EXXCGXXGXMXXDPXXLPEGVXXXXXRXCA; and
(e) a variant comprising a substitution, deletion, and/or insertion of one or several amino acids of the mature polypeptide of SEQ ID NO: 2.
In one further embodiment the endogenous polypeptide of the invention, the activity of which is reduced or eliminated in the mutant host, comprises the amino acid sequence of SEQ ID NO: 2 or an allelic variant thereof; or a fragment thereof. In a preferred aspect, the polypeptide comprises the amino acid sequence of SEQ ID NO: 2. In another preferred aspect, the polypeptide comprises the mature polypeptide of SEQ ID NO: 2. In another preferred aspect, the polypeptide comprises amino acids 20 to 79 of SEQ ID NO: 2, or an allelic variant thereof; or a fragment thereof. In another preferred aspect, the polypeptide comprises amino acids 20 to 79 of SEQ ID NO: 2. In another preferred aspect, the polypeptide consists of the amino acid sequence of SEQ ID NO: 2 or an allelic variant thereof; or a fragment thereof. In another preferred aspect, the polypeptide consists of the amino acid sequence of SEQ ID NO: 2. In another preferred aspect, the polypeptide consists of the mature polypeptide of SEQ ID NO: 2. In another preferred aspect, the polypeptide consists of amino acids 20 to 79 of SEQ ID NO: 2 or an allelic variant thereof; or a fragment thereof. In another preferred aspect, the polypeptide consists of amino acids 20 to 79 of SEQ ID NO: 2.
In a preferred aspect the mutant host cell is a filamentous fungal cell or a yeast cell. In a more preferred aspect the filamentous fungal host is selected from the group consisting of Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell.
Preferably the Aspergillus cell is selected from Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger or Aspergillus oryzae. 
In another preferred embodiment the mutant filamentous fungal cell is an Aspergillus oryzae cell or an Aspergillus niger cell.
Modification or inactivation of the nucleotide sequence may be performed by subjecting the parent cell to mutagenesis and selecting for mutant cells in which expression of the nucleotide sequence has been reduced or eliminated. The mutagenesis, which may be specific or random, may be performed, for example, by use of a suitable physical or chemical mutagenizing agent, by use of a suitable oligonucleotide, or by subjecting the DNA sequence to PCR generated mutagenesis. Furthermore, the mutagenesis may be performed by use of any combination of these mutagenizing agents.
Examples of a physical or chemical mutagenizing agent suitable for the present purpose include ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine, nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formic acid, and nucleotide analogues.
When such agents are used, the mutagenesis is typically performed by incubating the parent cell to be mutagenized in the presence of the mutagenizing agent of choice under suitable conditions, and screening and/or selecting for mutant cells exhibiting reduced or no expression of the gene.
Modification of the nucleotide sequence may be accomplished by introduction, substitution, or removal of one or more (several) nucleotides in the gene or a regulatory element required for the transcription or translation thereof. For example, nucleotides may be inserted or removed so as to result in the introduction of a stop codon, the removal of the start codon, or a change in the open reading frame. Such modification or inactivation may be accomplished by site-directed mutagenesis or PCR generated mutagenesis in accordance with methods known in the art. Although, in principle, the modification may be performed in vivo, i.e., directly on the cell expressing the nucleotide sequence to be modified, it is preferred that the modification be performed in vitro as exemplified below.
An example of a convenient way to eliminate or reduce expression of a nucleotide sequence by a cell is based on techniques of gene replacement, gene deletion, or gene disruption. For example, in the gene disruption method, a nucleic acid sequence corresponding to the endogenous nucleotide sequence is mutagenized in vitro to produce a defective nucleic acid sequence that is then transformed into the parent cell to produce a defective gene. By homologous recombination, the defective nucleic acid sequence replaces the endogenous nucleotide sequence. It may be desirable that the defective nucleotide sequence also encodes a marker that may be used for selection of transformants in which the nucleotide sequence has been modified or destroyed. In a particularly preferred aspect, the nucleotide sequence is disrupted with a selectable marker such as those described herein.
Alternatively, modification or inactivation of the nucleotide sequence may be performed by established anti-sense or RNAi techniques using a sequence complementary to the nucleotide sequence. More specifically, expression of the nucleotide sequence by a cell may be reduced or eliminated by introducing a sequence complementary to the nucleotide sequence of the gene that may be transcribed in the cell and is capable of hybridizing to the mRNA produced in the cell. Under conditions allowing the complementary anti-sense nucleotide sequence to hybridize to the mRNA, the amount of protein translated is thus reduced or eliminated.
The present invention further relates to a mutant cell of a parent cell that comprises a disruption or deletion of a nucleotide sequence encoding the polypeptide or a control sequence thereof, which results in the mutant cell producing less of the polypeptide or no polypeptide compared to the parent cell.
The polypeptide-deficient mutant cells so created are particularly useful as host cells for the expression of native and/or heterologous polypeptides. Therefore, the present invention further relates to a mutant cell according to the invention further comprising a gene encoding a native or heterologous protein and to methods of producing a native or heterologous polypeptide comprising: (a) cultivating the mutant cell under conditions conducive for production of the native or heterologous polypeptide; and (b) recovering the polypeptide. The term “heterologous polypeptides” is defined herein as polypeptides that are not native to the host cell, a native protein in which modifications have been made to alter the native sequence, or a native protein whose expression is quantitatively altered as a result of a manipulation of the host cell by recombinant DNA techniques.
In a further aspect, the present invention relates to a method of producing a protein of interest comprising: (a) cultivating a mutant cell under conditions conducive for production of the protein wherein the mutant has a reduced or no expression of an endogenous polypeptide compared to a parent host cell grown under the same conditions, and wherein the expression of said endogenous polypeptide commits the cell to apoptosis; and optionally (b) recovering the protein of interest.
In an even further aspect the present invention relatesto a method of producing a protein of interest, comprising: (a) cultivating the mutant cell having a reduced or no activity of the polypeptide of the invention under conditions conducive for production of the protein; and (b) recovering the protein. Particularly the protein is a heterologous protein.
The methods used for cultivation and purification of the product of interest may be performed by methods known in the art.
The methods of the present invention for producing a protein product are of particular interest in the production of eukaryotic polypeptides, in particular fungal proteins such as enzymes. The enzyme may be selected from, e.g., an amylolytic enzyme, lipolytic enzyme, proteolytic enzyme, cellulytic enzyme, oxidoreductase, or plant cell-wall degrading enzyme. Examples of such enzymes include an aminopeptidase, amylase, amyloglucosidase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, galactosidase, beta-galactosidase, glucoamylase, glucose oxidase, glucosidase, haloperoxidase, hemicellulase, invertase, isomerase, laccase, ligase, lipase, lyase, mannosidase, oxidase, pectinolytic enzyme, peroxidase, phytase, phenoloxidase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transferase, transglutaminase, or xylanase. The mutant cells may also be used to express heterologous proteins of pharmaceutical interest such as hormones, growth factors, receptors, and the like.
It will be understood that the term “eukaryotic polypeptides” includes not only native polypeptides, but also those polypeptides, e.g., enzymes, which have been modified by amino acid substitutions, deletions or additions, or other such modifications to enhance activity, thermostability, pH tolerance and the like.
Methods of Inhibiting Expression of a Polypeptide
In the production method according to the invention the reduction or elimination of the polypeptide according to the invention is particularly achieved by random mutation, site specific mutation, deletion, or by inhibiting the expression by administering a double-stranded RNA (dsRNA) molecule, wherein the dsRNA comprises a subsequence of a polynucleotide of the present invention. In a preferred aspect, the dsRNA is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more duplex nucleotides in length.
The dsRNA is preferably a small interfering RNA (siRNA) or a micro RNA (miRNA). In a preferred aspect, the dsRNA is small interfering RNA (siRNAs) for inhibiting transcription. In another preferred aspect, the dsRNA is micro RNA (miRNAs) for inhibiting translation.
The present invention also relates to such double-stranded RNA (dsRNA) molecules, comprising a portion of the mature polypeptide coding sequence of SEQ ID NO: 1 for inhibiting expression of a polypeptide in a cell. While the present invention is not limited by any particular mechanism of action, the dsRNA can enter a cell and cause the degradation of a single-stranded RNA (ssRNA) of similar or identical sequences, including endogenous mRNAs. When a cell is exposed to dsRNA, mRNA from the homologous gene is selectively degraded by a process called RNA interference (RNAi).
Methods for making and using dsRNA molecules to selectively degrade RNA are well known in the art, see, for example, U.S. Pat. No. 6,506,559; U.S. Pat. No. 6,511,824; U.S. Pat. No. 6,515,109; and U.S. Pat. No. 6,489,127.
The present invention is further described by the following examples that should not be construed as limiting the scope of the invention.
EXAMPLES
Chemicals used as buffers and substrates were commercial products of at least reagent grade.
Plasmids


    pDV8 is described in patent WO2001068864, example 8.
    pJaL410 is described in patent WO2003008575, example 8.
    pJaL419 is described in patent WO2003008575, example 8.
    pJaL504 is described in example 1 and SEQ ID NO: 14.
    pJaL554 is described in patent WO2001068864, example 8.
    pJaL575 is described in example 1 and SEQ ID NO: 15.
    pJaL729 is described in patent WO2003008575, example 12.
    pJaL731 is described in example 1 and SEQ ID NO: 16.
    pJaL736 is described in example 1 and SEQ ID NO: 17.
    pJAL958 is described in example 1 and SEQ ID NO: 18.
    pMT2786 is described in example 2 and SEQ ID NO: 19.
    pMT3155 is described in example 2 and SEQ ID NO: 20.
    pMT3296 is described in example 2 and SEQ ID NO: 21.
    pMT3306 is described in example 2 and SEQ ID NO: 22.
    pMT3328 is described in example 3 and SEQ ID NO: 23.
    pMT3330 is described in example 3 and SEQ ID NO: 24.
    pMT3332 is described in example 3 and SEQ ID NO: 25.
    pMT3369 is described in example 4 and SEQ ID NO: 26.
    pMT3376 is described in example 4 and SEQ ID NO: 27.
    pMT3378 is described in example 4 and SEQ ID NO: 28.
    pMT3431 is described in example 4 and SEQ ID NO: 29.

Strains

    ToC1512 is described in patent WO2005070962, example 11.
    JaL507 is described in example 1.
    BECh2 is described in WO 00/39322, example 1, which is further referring to JaL228 described in WO 98/12300, example 1.


Example 1
Identification of the ZY082808 Transcript
Construction of a Lipase Producing Strain of Aspergillus Oryzae 
Construction of the Aspergillus Oryzae niaD Deletion Plasmid pJaL736
The single restriction endonuclease sites BamHIand BgIII in pDV8 were removed by two succeeding rounds of cutting with each of the restriction endonucleases and the free overhang-ends was filled in by treatment with Klenow polymerase and the four deoxyribonucleotides and ligated resulting in plasmid pJaL504 (SEQ ID NO: 14).
From pJaL504 a 2514 bp fragment were amplified by PCR with primer 172450 and 172449 (SEQ ID NO: 3 and 4) and cloned into the vector pCR®4Blunt-TOPO resulting in plasmid pJaL575 (SEQ ID NO: 15).
The repeat flanked pyrG selection marker from pJaL554 was moved as a 1998 bp Asp718-HindIII fragment where the ends were completely filled in by treating with dNTP's and klenow polymerase. The fragment was cloned into completely filled in BssHII-Asp718 site of pJaL419 resulting in plasmid pJaL731 (SEQ ID NO: 16). The pyrG gene thereby replaces a 2200 bp fragment encoding the 3′ part of the niaD gene and the pyrG gene is then flanked by a 772 bp fragment of the 5′ end of niaD and a 698 bp fragment of the 3′ end of niaD. Finally, the deletion cassette of pJaL731 containing the two niaD flanks on either side of the pyrG selection marker was transferred as a 3500 bp HindIII fragment into the HindIII site of the tk counter selectable plasmid pJaL575 to give the deletion plasmid pJaL736 (SEQ ID NO: 17). Note that pJaL736 contains a unique NotI site, which were used to linearize the plasmid prior to transformation into A. oryzae. 
Construction of niaD Deleted A. Oryzae Strain, JaL507.
20 microgram of pJaL736 was cut with NotI and subsequently the enzyme was heat inactivated as recommended by the manufacturer (New England Biolabs). The plasmid was then ethanol precipitated and re-dissolved in Tris buffer (10 mM pH 8.0) at a concentration suitable for transformation into Aspergillus oryzae. 
The linearized plasmid DNA was transformed into Aspergillus oryzae ToC1512 with selection for pyrG and counter selection of the tk gene on FDU plates as previously described in WO 0168864. Transformants were re-isolated twice and finally grown up in liquid medium (YPD). Transformants were tested for there ability to grow on nitrate as the sole nitrogen source. Chromosomal DNA was prepared as previously described in WO 0168864 form transformants that were not able to grow on nitrate which indicate that the niaD gene was disrupted and used for Southern analysis of the niaD locus with the aim to identify transformants in which a clean double cross-over between the chromosomal niaD and the deletion cassette had occurred. The chromosomal DNA was digested with BamHI. The Southern blot was first probed with the whole niaD gene excised as a 3430 bp HindIII fragment from pJaL410. For the intact niaD locus, two bands of 2838 bp and 1820 bp respectively was appearing using this probe, while the niaD deleted derivative originating from the desired double cross-over resulted in a 4567 bp band. The Southern was stripped of the first probe and re-probed with a probe excised as a 2059 bp Asp718 fragment of pJaL410. For the intact niaD locus a 2838 bp and a 1820 bp BamHI band were appearing, while for the niaD deletion strain originating from the desired cross over no hybridization bands was obtained. A strain with the above characteristics was preserved as A. oryzae strain JaL507.
Construction of pJaL958
A variant of the Humicola lanuginosa lipase was cloned as a 901 bp BamHI-SaII obtained from digestion of p960 (described in EP0305216 example 1 and FIG. 4) into pJaL729 digested with BamHI-XhoI resulting in pJaL958 (SEQ ID NO: 18).
Construction of A. Oryzae Strain JaL731
Plasmid pJaL958 was transformed into A. oryzae strain JaL507 and transformants were screened for lipase activity as described in WO2003008575, example 13-15. Among the transformants one displaying high lipase activity was selected and named JaL731.







Primer 172450


(SEQ ID NO: 3)


5′-GACGAATTCTCTAGAAGATCTCTCGAGGAGCTCAAGCTTCTGTA-


CAGTGACCGGTGACTC


 


Primer 172449


(SEQ ID NO: 4)


5′-GACGAATTCCGATGAATGTGTGTCCTG






Spores of JaL731 were subjected to mutagenesis using nitrosoguanidine (as described in Araujo, Ward and D'Souza. (1991). Biotechnology Techniques 5 (4): 283-288. Use of mutation strategies applied to Aspergillus terreus ATCC 52430 to obtain mutants with improved cellulase productivity). Mutants were screened for increased lipase expression in microtiter plates by using robotics, and a high yielding mutant, named 5-58, was selected. Lipase activity was measured as described by Haack et al., 2006 (Change in hyphal morphology of Aspergillus oryzae during fed-batch cultivation. Appl Microbiol Biotechnol 70: 482-487).
The two lipase producing strains, JaL731 and its high yield mutant 5-58, were compared by transcriptomic analysis of gene expression by using DNA microarray technique (Baldi and Hatfield, 2002 (DNA Microarrays and Gene Expression, Cambridge University Press, Cambridge); Knudsen, 2002 (A Biologist's Guide to Analysis of DNA microarray data. Wiley & sons, New York).
Spotted slides based on an inhouse cDNA library of A. oryzae expressed sequence tags were used. mRNA was extracted by the PolyATract® System 1000 (Promega Corporation, Madison, Wis.), starting with 1 g of cell material. cDNA synthesis and Cy3 and Cy5 labelling were performed with the aid of the CyScribe Post labelling Kit with CyScribe GFX purification kit (Amersham Biosciences, HiHerod, Denmark). Hybridization was performed using standard methods. Scanning of the slides was performed using a GMS 418 array scanner (Genetic MicroSystems Inc., Woburn, Mass.). The scanning images were quantified by using the ImaGeneTM 5 Software (Biodiscovery Inc., Marina Del Rey, Calif., USA). Statistical analysis of differential expression was performed using Genesight Software (Biodiscovery Inc., Marina Del Rey, Calif., USA).
Laboratory tank fermentation was carried out for each of the two strains JaL731 and 5-58, and sampling of biomass was done at two time points. Eight successful hybridizations were carried out between the two strains. The transcriptomic analysis showed differential regulation of the transcript ZY082808. In the high yield mutant, 5-58, the ZY082808 transcript was down-regulated in.
A number of genes were differentially regulated. However, also microarray data from analyses of other A. oryzae lipase strain pairs suggested ZY082808 as an interesting gene for follow up. Thus, gene expression of a high producing recombinant lipase strain Amsp173-40a was compared to an analogous lower producing recombinant lipase strain Amsp165-28a by using the DNA microarray technique. The strain Amsp173-40a is described by Haack et al. (2006) supra. Amsp165-28a was constructed in the same way as Amsp173-40a and selected as a low producing strain by test in laboratory tank fermentation. For Amsp173-40a increased swelling of the hyphal tip was observed in the fed batch phase with high lipase productivity and assumed secretion stress conditions as described and discussed by Haack et al. (2006) supra. For Amsp165-28a no swelling of the hyphal tip was observed. The transcriptomic analysis showed differential regulation of ZY082808 also in these strains, and ZY082808 was up-regulated in Amsp173-40a in a number of independent array experiments as shown in table 1. The reverse regulation of ZY082808 in the two different strain pairs suggested ZY082808 as a particular interesting gene for follow up.











TABLE 1








Amsp173-40a
Amsp173-40a
Amsp173-40a
5-58



 
 
 
 



Amsp165-28a
Amsp165-28a
Amsp165-28a
JaL731



Exp1
Exp. 2
Exp. 3
Exp. 4
















Fold Change
P value
Fold Change
P value
Fold Change
P value
Fold Change
P value





ZY
2.8
 7.7
2.8
 6.3
3.2
 4.9
−3.1
<


082808

10−6

10−5

10−5

0.001









Example 2
Expression Plasmids for Overexpression of the ZY082808 Encoded Peptide
Primers:








Primer 821
(SEQ ID NO: 5)


5′-TCTTGGTACCCTCAGCAAAGCCATCATTGC-3′



 


Primer 822
(SEQ ID NO: 6)


5′-TCTCTCTAGACTCCCTCTGGTT-



TAATACTCCG-3′



 


Primer 829
(SEQ ID NO: 7)


5′-CATTTGGATCCACCATGAGACTCTCCC-3′



 


Primer 841
(SEQ ID NO: 8)


5′-TTCTCTCGAGCTAAGGAAACCACCTCGGC-3′







The A. oryzae chromosomal sequence encoding the ZY082808 transcript was PCR amplified from A. oryzae strain BECh2 using primers 821 and 822 (SEQ ID 5 and 6) to give a 2.0 kb fragment. This fragment was cut at the Asp718 and Xba1 sites introduced by the applification primers 821 and 822. The ZY082808 encoding Asp718-Xba1 fragment was inserted in the vector pMT3155 (SEQ ID NO: 20) which had been cut with BsiW1 and Bln1. The pMT3155 vector contains markers to allow for selection of leucine prototrophy in E. coli and for selecting resistance to the herbicide BASTA in Aspergillus oryzae. The resulting plasmid capable of directing expression of the ZY082808 encoded peptide from its own original promoter was named pMT3296 (SEQ ID NO: 21).
To construct a plasmid expressing the ZY082808 encoded peptide from a strong tandem NA2/TPI promoter described in WO 2003008575, the ZY082808 ORF was PCR amplified from pMT3296 using primers 829 and 841 (SEQ ID NO: 7 and 8) to give a 0.25 kb fragment.
This fragment was cut at the BamH1 and Xho1 sites introduced by the PCR primers, and inserted in the vector pMT2786 (SEQ ID NO: 19) (which is a modified version of pMT2188 described in WO 2006/050737 example 2; in pMT2786 the URA3 marker has been replaced with the S. cerevisiae LEU2 marker which can be selected for in E. coli leuB mutants (leucine requiring), and in addition there is an approximately 2 kb “stuffer” inserted in pMT2786 between the BamH1 and Xho1 used for cloning) cut with Bam and Xho. The pMT2786 vector contains markers to allow for selection of leucine prototrophy in E. coli and for selecting for ability to grow on acetamide as sole nitrogen source in A. oryzae. The resulting expression plasmid was named pMT3306 (SEQ ID NO: 22).
pMT3296 was transformed into A. oryzae mutant strain 5-58 selecting for resistance to BASTA. 12 transformants were isolated and subsequently re-isolated twice through sporulation. Likewise, pMT3306 was transformed into strain 5-58 selecting for the ability to grow on acetamide as sole nitrogen source. No transformants were obtained growing detectably better than the background untransformed A. oryzae strain. The transformation experiment was repeated but again no transformants could be isolated.
The 12 transformants as well as the strain 5-58 were inoculated on Cove-N agar slants and incubated for one week at 34° C. For all isolates 500 ml shake flasks with 2 baffles and with 100 ml G1-Gly substrate was inoculated with 5 ml spore suspension and incubated over night at 30° C., 250 rpm. The G1-Gly substrate contained 18 g/l yeast extract, 24 g/l glycerol 87%, 1 ml/l pluronic and 5 g/l CaCO3. Tapwater was used. pH was adjusted to 7.0 before sterilization for 20 min at 121° C. From each G1-Gly shake flask 5 ml was transferred to two 500 ml shake flasks with 2 baffles and with 100 ml 1/5MDU2B substrate. The 1/5MDU2B substrate contained 9.0 g/l maltose, 0.2 g/l MgSO4.7H2O, 0.2 g/l NaCl, 0.4 g/l K2SO4, 2.4 g/l KH2PO4, 0.1 ml/l trace metal, 0.02 ml/l pluronic, and 1.4 g/l yeast extract. Tapwater was used. The trace metal solution contained 14.2 g/l ZnSO4.7H2O, 2.5 g/l CuSO4.5H2O, 0.5 g/l NiCl2.6H2O, 13.8 g/l FeSO4.7H2O, 8.5 g/l MnSO4.H2O and 3.0 g/l citric acid H2O. pH was adjusted to 5.0 before sterilization for 20 min at 121° C. After sterilization 0.2 ml 50% urea was added pr. 100 ml substrate. The 1/5MDU2B shake flasks were incubated at 34° C., 270 rpm. Every shake flask was sampled after 2 days. Samples were centrifuged for 10 min at 3000 rpm. Supernatants are frozen at −20° C. Samples were thawed and lipase activity (LU/ml) was measured by using standard protocol. Two independent shake flaks experiments were carried out.
Four out of twelve transformants had reproducibly 2-3 times reduced lipase expression compared to 5-58 as shown in table 2. This indicates a negative effect of over-expression of ZY082808 as suggested by transcriptomic analysis. It can be seen that for e.g. transformant 5-58pMT3296#3 an increased expression was observed, which could be due to some random variation, i.e. copy number increase of the lipase gene combined with low expression of ZY082808 in that particular transformant. The results should be compared to transformation by the vector alone. Twenty pMT3155-transformants were fermented by using the same protocol as for the 5-58pMT3296 transformants.
No significant negative effect on lipase expression was observed for the twenty transformants as shown in table 3. This confirms the suggested negative effect of ZY082808 overexpression from the data shown in table 2.










TABLE 2







Experiment 1
Experiment 2




Lipase expression in
lipase expression in



Strain
% of 5-58
% of 5-58



















5-58pMT3296#1
126
109



5-58pMT3296#2
114
115



5-58pMT3296#3
138
140



5-58pMT3296#4
36
37



5-58pMT3296#5
27
33



5-58pMT3296#6
98
104



5-58pMT3296#7
33
38



5-58pMT3296#8
113
112



5-58pMT3296#9
144
119



5-58pMT3296#10
118
113



5-58pMT3296#11
93
87



5-58pMT3296#12
55
55



5-58
100
100



















TABLE 3







Experiment 1
Experiment 2




lipase expression in
lipase expression in



Strain
% of 5-58
% of 5-58



















5-58
100
100



5-58pmt3155#1
74
98



5-58pmt3155#2
83
76



5-58pmt3155#3
73
114



5-58pmt3155#4
52
112



5-58pmt3155#5
75
102



5-58pmt3155#6
ND
95



5-58pmt3155#7
ND
129



5-58pmt3155#8
ND
110



5-58pmt3155#9
ND
101



5-58pmt3155#10
ND
ND



5-58pmt3155#11
ND
96



5-58pmt3155#12
ND
106



5-58pmt3155#13
ND
124



5-58pmt3155#14
ND
126



5-58pmt3155#15
80
107



5-58pmt3155#16
94
102



5-58pmt3155#17
68
98



5-58pmt3155#18
70
104



5-58pmt3155#19
106
77



5-58pmt3155#20
78
105









Example 3
Construction of a Plasmid for Expression of a shRNA for ZY82808 Sequences
In order to have a visual marker by which to judge the degree of downregulation, a construct was made in which both ZY082808 and the wA gene needed for green spore development could be downregulated. Absence of the wA gene product leads to white spore color, and partial expression of the gene could be expected to lead to lighter shades of green than wt spore color.
Primers:









Primer 798



(SEQ ID NO: 9)



5′-TCTTGGATCCGTCGACCTTGGGGAAGTCATCACCG-3′ 


 



Primer 868



(SEQ ID NO: 10)



5′-GTCATTAATCATTGAATTCTCGTCGCGACTCTC-3′ 


 



Primer 865



(SEQ ID NO: 11)



5′-GTCGCGACGAGAATTCAATGATTAATGACCTTTTC-



CTAAATATAC-3′ 


 



Primer 867



(SEQ ID NO: 12)



5′-TCTTCTCGAGCCTAGGTATCCCAAAGTCAA-



GTACGTCAGG-3′


 



Primer 866



(SEQ ID NO: 13)



5′-TCTTCTCGAGCCTAGGCTCTCCCTCAACCTTCTACTG-3′ 






A fusion of the sequences encoding part of the wA (whiteA) product (Thomas et al., 1998, Microbiol and Mol Biol Reviews, 62(1): 35-54) and the ZY082808 sequence including part of the 5′UTR was made by SOE PCR: The wA sequences were amplified from A. oryzae strain BECh2 DNA using primers 798 and 868 (SEQ ID NO: 9 and 10, respectively) and fused to ZY082808 sequences amplified by primers 865 and 867 (SEQ ID NO: 11 and 12) from pMT3296 template. The SOE fusion product was cut by BamH1 and Xho1 at the sites introduced by primers 798 and 867, respectively. The resulting approx. 1.2 kb fragment was ligated into BamH1-Xho1 cut vector pMT2786 to give plasmid pMT3328 (SEQ ID NO: 23).
A similar fusion of wA to a shorter piece of ZY082808 sequence (lacking 5′UTR) was made by fusing the wA fragment amplified by primers 798 and 868 to the ZY082808 sequence amplified by primers 865 and 866 (SEQ ID NO: 11 and SEQ ID NO: 13). The resulting approx. 1.0 kb fragment was cut by BamH1 and Xho1 at the sites introduced by primers 798 and 866, respectively. The Bam-Xho fragment was inserted in BamH1-Xho1 cut vector pMT2786 to give plasmid pMT3330 (SEQ ID NO: 24). Finally the Asp718-Xho1 (5.5 kb) and the Asp718-Bln1 (2.5 kb) fragments of pMT3328 were ligated to the Bln1-SaI1 (1.0 kb) fragment to give plasmid pMT3332 (SEQ ID NO: 25). pMT3332 can be selected in Aspergillus oryzae by the ability to grow on acetamide as the sole source of nitrogen. pMT3332 is constructed such that the strong NA2/TPI promoter drives the transcription of a shRNA containing sequences from both wA and ZY082808. Transformants with pMT3332 will thus contain double stranded RNA for both wA and ZY082808 and expression of both wA and ZY082808 is expected in various degrees to be knocked down in A. oryzae strains transformed with pMT3332.
pMT3332 was transformed into A. oryzae strain JaL731 and 24 transformants selected by their ability to grow on acetamide as sole source of nitrogen. Transformants were twice reisolated as described above.
The 24 transformants were evaluated in two independent shake flask experiments. As shown in table 4, five to ten of the transformants seemed to have 10-25% increased lipase expression compared to JaL731 although there is some uncertainty due to a high variation from experiment to experiment. This is in line with the clear negative effect of over-expression of ZY082808 in 5-58.
In summary both microarray data for various independent lipase strains, follow up studies by over-expression and by attempted down regulation by shRNA show, that ZY082808 has significant influence on lipase expression.










TABLE 4







Experiment 3
Experiment 4




Lipase expression
Lipase expression




in
in



Strain
% of JaL731
% of JaL731



















JaL731pMT3332#1
125
ND



JaL731pMT3332#2
106
ND



JaL731pMT3332#3
123
124



JaL731pMT3332#4
126
128



JaL731pMT3332#5
105
105



JaL731pMT3332#6
113
106



JaL731pMT3332#7
133
124



JaL731pMT3332#8
125
ND



JaL731pMT3332#9
101
105



JaL731pMT3332#10
ND
ND



JaL731pMT3332#11
106
ND



JaL731pMT3332#12
89
114



JaL731pMT3332#13
108
102



JaL731pMT3332#14
113
ND



JaL731pMT3332#15
121
119



JaL731pMT3332#16
ND
ND



JaL731pMT3332#17
131
120



JaL731pMT3332#18
139
157



JaL731pMT3332#19
126
163



JaL731pMT3332#20
50
ND



JaL731pMT3332#21
94
ND



JaL731pMT3332#22
118
166



JaL731pMT3332#23
72
56



JaL731pMT3332#24
123
95



JaL731
100
100









The invention described and claimed herein is not to be limited in scope by the specific aspects herein disclosed, since these aspects are intended as illustrations of several aspects of the invention. Any equivalent aspects are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the case of conflict, the present disclosure including definitions will control.
Example 4
Construction of a ZY082808 Deleted A. Oryzae Strain MT3420 and Expression of a Variant Humicula Lanuginose Lipase
First a ZY082808 deleted A. oryzae strain, MT3420, was constructed. The sequences flanking the ZY082808 ORF were PCR amplified with primers based on the published A. oryzae genomic sequence (Machida, M. et al. Nature 438 (2005)1157-1161) using genomic DNA from A. oryzae strain BECh2 as template. The 5′ flank was amplified using primers 905 and 904 (SEQ ID NO: 31 and 30) to give a PCR fragment of 1162 bp, whereas the 3′flank was amplified by primers 907 and 906 (SEQ ID NO: 33 and 32) to give an 1150 bp fragment. Primers were designed to allow subsequent SOE PCR using primers 905 and 906 to join the fragments placing a Hind3 site between the two flanks. The SOE assembled fragment (2.2 kb) was cloned into the pCR4 TOPO blunt (InVitrogen) to give pMT3369 (SEQ ID NO: 26). The repeat flanked pyrG selection marker was inserted as a Hind3-Hind3 fragment (as described in example 1) into the unique Hind3 site of pMT3369 to give plasmid pMT3376 (SEQ ID NO: 27). Finally the deletion cassette of pMT3376 containing the two flanks of ZY082808 on either side of the pyrG selection marker was transferred as a Not1-Spe1 fragment into the Not1-Xba1 sites of the tk counterselectable plasmid pJaL504 to give the deletion plasmid pMT3378 (SEQ ID NO: 28). Note that pMT3378 contains a unique Not1 site immediately downstream of the 3′ flank. This can be used to linearize the plasmid prior to transformation into A. oryzae. 
The linearized plasmid pMT3378 was transformed into A. oryzae JaL355, as described in WO2007045248, by selection for pyrG and counter selection for the tk gene on FDU plates as previously described. 34 transformant colonies were twice reisolated and finally grown up on liquid medium (YPD). Chromosomal DNA was prepared and used in the first place for PCR analysis of the ZY082808 locus. Primers 995 and 996 (SEQ ID NO: 34 and 35) were used for the amplification. The amplified band is expected to have a size of 3.32 kb in the wt strain, while a band of 5.89 kb is expected when the pMT3378 derived deletion cassette has been inserted. Seven of the 34 selected colonies showed the band expected for the deletion strain.
Four potential deletion strains were chosen for Southern verification of the deletion. DNA from a non-deleted control A. oryzae strain was run in parallel. The genomic DNA was digested with either BamH1 or with BgI2 and probed with a PCR fragment generated from primers 999 and 1000 (SEQ ID NO: 36 and 37) (A. oryzae BECh2 template). In the BamH1 digested samples of the control strain the probe hybridized to the expected size band of 3.9 kb while three out of the four potentially ZY082808 deleted strains showed the predicted bands of 2.0 kb and 1.35 kb. In the BgI2 digest the hybridizing bands were 4.7 kb for the control and 6.4 kb plus 0.9 kb for the same three potentially deleted strains. The fourth deletion candidate strain showed a more complex pattern presumably due to a rearrangement during the integration event. This fourth strain was discarded. Two of the strains showing a clean ZY082808 disruption in the Southern were frozen to the strain collection as MT3420 and 3421.
MT3420 was transformed with pMT3431 (SEQ ID NO: 29) selecting for the amdS marker. pMT3431 is an expression plasmid for the variant Humicola lanuginose lipase described under construction of pJaL958, example 1. The main difference between pJaL958 and pMT3431 is the marker for selection which in pMT3431 is the A. nidulans amdS gene.
Transformants were obtained and twice reisolated through sporulation. The reisolated transformants were grown and tested for their expression level of the lipase variant.
Transformants of the parent strain A. oryzae Bech2 were obtained and tested for parallel comparisons.
Shake Flask Evaluation
Two times ten lipase transformants made in respectively MT3420 and BECh2 were tested in shake flask experiments by measurement of lipase activity (LU/ml). The same set up as described in example 2 was applied except that both 1/5MDU2B and 1/5GDU2B shake flask medium were used for expression. 1/5GDU2B is composed as 1/5MDU2B apart from maltose has been substituted with glucose.
In the first experiment the series of transformants in MT3420 had an approx. 20% improved expression in maltose fermentations judged by average and median compared to the series in BECh2 as shown in Table 5. No difference between the hosts was observed in glucose fermentations, meaning that the maltose/glucose expression ratio was increased for transformants in MT3420.
Also in the second shake flask experiment a somewhat increased maltose/glucose expression ratio was observed for the series of transformants in MT3420 compared to the series in BECh2. The experiments have been hampered of variation originating from the none-robust lipase analysis and the nature of the transformants.
In conclusion, there is indication of improved performance for the host MT3420.







TABLE 5







Expression of 10 transformants in MT3420 and 10 transformants in


BECh2 ranked after normalization proportionate to the highest


expressing transformant in 1/5MDU2B in two independent experiments.












1/5MDU2B

1/5 GDU2B













MT3420
BECH2
MT3420
BECH2












Experiment 1











1
100
68
150
141


2
92
60
128
141


3
69
56
122
130


4
61
53
109
121


5
59
52
96
91


6
51
52
95
87


7
45
44
89
83


8
45
34
71
65


9
40
33
41
25


10
33
26
38
8









Experiment 2











1
85
100
86
128


2
77
98
85
109


3
75
95
82
102


4
73
82
80
72


5
71
75
75
65


6
58
39
62
65


7
57
38
60
58


8
52
34
53
35


9
51
21
44
19


10
40
7
23
5

## Claims

1. A mutant fungal host, wherein the mutant has a reduced or no expression of an endogenous polypeptide selected from the group consisting of:
(a) a polypeptide comprising an amino acid sequence having at least 90% identity to the mature polypeptide of SEQ ID NO: 2;
b) a polypeptide encoded by a polynucleotide comprising a nucleotide sequence having at least 90% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1.

2. The mutant according to claim 1, wherein the endogenous polypeptide comprises the mature polypeptide of SEQ ID NO: 2.

3. The mutant according to claim 2, wherein the endogenous polypeptide consists of the mature polypeptide of SEQ ID NO: 2.

4. The mutant according to claim 3, wherein the endogenous polypeptide is encoded by a polynucleotide which comprises or consists of the nucleotide sequence of SEQ ID NO: 1.

5. The mutant according to claim 4, wherein the endogenous polypeptide is encoded by a polynucleotide which comprises or consists of the mature polypeptide coding sequence of SEQ ID NO: 1.

6. The mutant according to claim 2, wherein the mature endogenous polypeptide is amino acids 20 to 79 of SEQ ID NO: 2.

7. The mutant according to claim 1, wherein the mature endogenous polypeptide coding sequence is nucleotides 58 to 237 of SEQ ID NO: 1.

8. The mutant fungal host according to claim 1, wherein the fungal cell is a filamentous fungal cell.

9. The mutant fungal host according to claim 8, wherein the filamentous fungal host is selected from the group consisting of Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, and Trichoderma cell.

10. The mutant fungal host according to claim 9, wherein the Aspergillus cell is selected from the group consisting of Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger and Aspergillus oryzae.

11. The mutant cell claim 1, further comprising a gene encoding a native or heterologous protein.

12. A mutant Aspergillus host cell, wherein the mutant has a reduced or no expression of an endogenous polypeptide selected from the group consisting of:
(a) a polypeptide comprising an amino acid sequence having at least 90% sequence identity to the mature polypeptide of SEQ ID NO: 2;
(b) a polypeptide encoded by a polynucleotide comprising a nucleotide sequence having at least 90% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1.

13. The mutant host cell of claim 12, wherein the host cell is Aspergillus oryzae and the polypeptide comprises an amino acid sequence having at least 95% sequence identity to the mature polypeptide of SEQ ID NO: 2.

14. The mutant host cell of claim 12, wherein the host cell is Aspergillus oryzae and the polypeptide comprises an amino acid sequence having at least 99% sequence identity to the mature polypeptide of SEQ ID NO: 2.

15. The mutant host cell of claim 12, wherein the host cell is Aspergillus oryzae and the polypeptide consists of the mature polypeptide of SEQ ID NO: 2.